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Beamtime guide - Infrared microspectroscopy
Infrared microspectroscopy

Beamtime application guide - Infrared microspectroscopy

What to include in proposal details

Users applying for the first time, or submitting a proposal for a new experiment must contact a member of the IRM group to discuss the experiment prior to submitting their proposal.  For helpful information regarding applications, access type, proposal deadlines and beamtime schedules please visit this link.

Users should include the following details in their experiment proposal:

  • Details of any previous lab-based IR measurements they have undertaken on these samples

  • Summary of outcomes from any previous synchrotron IR beamtime relating to these samples

  • Description of why the experiment requires use of the synchrotron beamline

  • How the samples will be prepared and mounted for analysis

  • Proposed experimental configuration (e.g. reflection, transmission, ATR, grazing angle, narrow-band MCT detector, rapid scan acquisition)

  • Estimation of time required per sample and therefore overall beamtime required (3 shifts available/day). Please see below FAQs for information on estimating the time required to map your sample

  • Any specific conditions for the sample environment (e.g. use of the Linkam sample stage)

  • Possible requirements for concurrent use of the FPA microscope

  • Requirements for specific equipment or access to onsite laboratories (e.g. Chemistry lab, pellet press, PC2 Biochemistry lab)

 Also please remember that the IRM beamline and the THz/Far IR beamline are two separate beamlines that perform very different experiments.

Beamline-specific admin procedures

A beamline induction is given by IR beamline staff to all members of a user group at the start of their experimental run, and is required to be renewed yearly. This induction is in addition to the Facility User Safety Training undertaken by all users. No user on the IR Microscope beamline may operate equipment or use the synchrotron facilities until they have completed both inductions. All users listed on an experiment authorisation (EA) form should attend at the beginning of an experimental run to complete the beamlines induction.

If you require access to the chemistry, biochemistry or cell culture labs, you will need to attend a separate induction to the beamline specific one.  These occur at 9:30 am Monday to Friday, or can be arranged at an alternative time if required.  These are run by the Lab Manager, Clare Scott (

Frequently Asked Questions

What wavelengths can this beamline cover?

Our FTIR microscope typically operates in the mid-IR region from 750 cm-1 up to 3800 cm-1. The long wavelength range can also be extended to 550 cm-1 if required using the wide band detector but with a 10x loss in sensitivity, and a larger aperture size is required, compromising spatial resolution (figures 11 and 12). We recommend that this detector only be used if you are interested in specific bands within the 900-550 cm-1 range. Please note that the spectra below were acquired with the older blade apertures, but demonstrate the point.  We now use circular apertures that can yield a focused beam size ranging from approx. 1.17 to 20.83 µm.

 Using a 20x20 μm2 aperture. 	Using a 5x5 μm2 aperture.
Figure 11. Extension of the lower wavenumbers obtainable using a wideband detector and a 20x20 μm aperture


Using a 5x5 μm2 aperture
Fig 12. Extension of lower wavenumbers not obtained using wideband detctor with 5x5 µm aperture due to loss of sensitivity

How do I demonstrate that I need the synchrotron for my experiments?

When preparing your beamtime proposal, it is important that you demonstrate that there is a genuine need for the high lateral spatial resolution achievable at the beamline.  The beam can be focused to a spot size ranging from ~1-20 µm, depending on the experimental configuration and is ideal for rastor mapping complex heterogeneous polymeric samples, single cell, or complex biological tissue, for example. Ideally, you will specifically state the spatial resolution that you required, in microns. Remember that as the wavelength of mid-IR radiation is in the 3-10 µm range, this will thereby essentially limit the smallest sized objects that can be individually resolved using this beamline.  The size of the beam, according to the sampling mode as well as the objective and pinhole used, can be estimated from Figure 13, under the tab below (How long will it take to measure an IR map of my sample?).

Experiments requiring a spot size of 20 µm or greater, or that require 'bulk' or average measurements per sample, will have no advantage using the synchrotron and can be carried out on a lab-based IR microscope. However, If you can show that desired results were unsuccessful using a lab-based IR microscopy, this can be advantageous for your application to use our beamline. Furthermore, we now offer time resolved IRM, or rapid scan analysis, with the ability to measure up to 65 spectra/sec at a resolution of 16 cm-1, and a scanner velocity of 160 kHz.  Requirements of the time resolved spectroscopy could justify the requirement of the synchrotron IRM beamline without the need for spatial resolution.  Please contact beamline staff if you are unsure whether your experiment would benefit from use of the synchrotron.

How long will it take to measure an IR map of my sample?

When preparing your beamtime application, the amount of time that you apply for will depend on the number of samples you have and the amount of time required to map each one.  It is helpful if you can clearly outline your specific time requirements in your application.

The time taken to map an area on your sample can be estimated based on the area that you would like to map, the step size (distance between each spectra/pixel) and the time take to acquire each spectral point in your map.  Information on the beam size according to experimental set up is provided in figure 13 below, which shows the dependence on the sampling mode, objective and manual pinhole used.  This information, along with the time taken per spectra (based on the scan averaging number), allows you to estimate the time required to map a sample.  The time calculated per spectra in Figure 13, is based on a spectral resolution of 4 cm-1. Two calculations are provided below, with examples for both transmission and macro-ATR modes.

Example map calculation in transmission mode:

In transmission mode, using the 36 X objective and a manual pinhole of 0.2, the size of the focused beam at your sample is 5.56 µm.  If you wanted to map a 200 x 200 µm area with an x and y  step size of 5 µm, the total number of spectra in the map would be ((200/5) x (200/5)) 1600. If you averaged each spectra from 32 scans at a spectral resolution of 4 cm-1, the time would be calculated by:

(1600 * 9 seconds)/60 = 240 minutes  (4 hours)

Example map calculation using macro-ATR:

Generally we recommend that for each sample, you acquire a low resolution overview map of your sample, before focusing on a region of interest at higher resolution, while maintaining the single contact between the ATR crystal and your sample.  This allows us to observe regions of specific interest of your sample as well as regions of best contact between the crystal and your sample.  Therefore for each sample, estimate the time required for both the low and high resolution map: 

Low resolution overview map:

In macro-ATR mode using a manual pinhole of 0.3, the beam size on your sample is 3.13 µm. However, for the "low resolution map" we could use an x and y  step size of 10 µm over a 200 x 200 micron area.  That gives a total of 400 points (spectra) in the map, as calculated by ((200/10) x (200/10)). Obtaining spectra averaged from 16 scans, approximates 6 seconds per spectra ( at 4 cm-1 resolution).  Therefore the total map time is calculated as follows:

High resolution overview map:

Without changing the contact between the sample and the ATR crystal, a high resolution map can be performed based on the information obtained from the above overview map.  For example, an area of 120 x 120 µm will be mapped at an x and y step size of 3 micron, with each spectra averaged from 16 scans, the time taken is:

((120/3)x (120/3) x 6)/60 = 160 minutes (1 hour and 40 minutes)

Therefore, considering both the high and low resolution maps you would estimate approximately 2.5 hours of mapping per sample .

Map times can be estimated from the beamsize and time taken per scan (pixel)
Fig 13. Map times can be estimated from the beamsize and time taken per spectra (at a resolution of 4 cm-1)


What exactly is the difference between the THz/Far IR beamline and the IR Microscopy beamline?

The IR Microspectroscopy beamline operates in the mid-IR region (typically 750-3800 cm-1) and is ideal for studying condensed-phase, heterogeneous samples due to the high lateral spatial  resolution that can be achieved. The measurement area can be decreased to diffraction-limited spot sizes of around 3-5 µm , and the Bruker microscope stage can step across a sample in precise steps, less than 1 µm. This beamline is thus ideal for creating chemical maps of a sample by rastor mapping along complex samples over regions of interest.

The THz/Far IR beamline operates at much longer wavelengths, down to 10 cm-1. At these longer wavelengths the larger minimum spot size means this beamline does not perform the same type of spatial mapping across a sample. Instead this beamline takes advantage of the synchrotron source and their Bruker spectrometer to measure:

  • Gaseous samples at high spectral resolution, by which we mean the beamline can measure extremely detailed IR spectra of gases by stepping the IR wavelength in precise increments of down to 0.00096 cm-1, &

  • The THz spectrum of condensed phase, homogenous samples, by placing the sample in the beam path where the beam has a typical spot size of about 3 mm. Here, as with IRM condensed phase samples, there is no advantage to going to high spectral resolutions so samples are typically scanned at spectral resolutions of between 4-8 cm-1

Please speak to beamline scientist if you would like to conduct experiments across both beamlines. Each beamline requires its own, separate proposal application for beamtime.

Can I measure the depth profile of my sample?

This beamline cannot do depth profiling (axial mapping of a sample), however when operating in transmission mode the z-height of the microscope stage can be moved through the focus of your sample in a set step size.  So if your sample is thicker than 5 µm, e.g. you are working with a 30 µm polished wafer of rock with an inclusion in the middle, you can focus the beam into your inclusion. The lateral spatial resolution achievable does degrade from the diffraction limit however when you have thicker samples.

The ATR on our beamline is also of a fixed angle, so again cannot be used to profile depth.

You recommend use of transmission over transflection IR spectroscopy where possible. Why is that?

IR analysis can be run using transflection, which requires sections to be laid onto IR reflective slides e.g. Kevley MirrIR slides or ITO coated glass, instead of straight transmission. MirrIR slides are much cheaper than IR transmissive slides such as calcium fluoride and have the larger dimensions of normal microscope slides (1 x 3 inches), so you can potentially lay 1-3 sections per slide. There are inherent problems with this method however, in that your sample e.g. if working with tissue sections, have to be extremely uniform and ideally 4 µm thick, rather than the 5-8 µm thick required for transmission with a bit more leeway in the uniformity. Results are also more difficult to interpret using this method. We recommend the following papers discussing the potential issues with transflection analysis for further information.

P. Bassan, J. Lee, A. Sachdeva, J. Pissardini, K.M. Dorling, J.S. Fletcher, A. Henderson, P. Gardner. Analyst, 138, 144 (2013)

K. Malek, B.R. Wood, K.R. Bambery. FTIR Imaging of Tissues: Techniques and Methods of Analysis; Chapter 15. in book M. Baranska (ed.), Optical spectroscopy and computational methods in biology and medicine. Springer Science, 2014.

M. Miljkovic, B. Bird, M. Diem. Analyst, 137, 3954 (2012)

Do I need to microtome my samples for analysis?

The synchrotron has a microtome onsite that you can access if requested as part of your beamtime. The microtome is used for samples that are in the form of a solid plastic-like material, or that can be embedded in an IR transmissive material like AgCl e.g. using a pellet press (the synchrotron also has a pellet press onsite). Microtoming involves taking transverse slices of the embedded sample into pieces ideally 5 µm thick (perhaps thinner if they are strongly absorbing) for analysis in transmission. In addition, if you have samples for use with the ATR, the microtome can be used to expose a smooth surface for analysis.

Site access can be granted earlier than your scheduled beamtime to prepare microtomed samples if arranged with beamline staff prior to your beamtime.

If you require microtomed tissue and/or cryosections, or your samples are tricky and you require expert help, we recommend contacting the Monash Histology Platform for advice.

Which IR transmissive windows should I use for my experiment?

This depends on the properties of your samples and the wavenumber region of interest. Different window materials will transmit different wavelengths to different extents in the mid-IR. We typically purchase our windows from Crystran, a company based in the UK, and recommend use of calcium fluoride or barium fluoride windows at 0.5 mm thick, or zinc selenide windows at 1 mm thick (due to issues with fringing across the baseline of spectra at 4 cm-1 spectral resolution using 0.5 mm thick windows). Ideally, the diameter of the window should be between 12 - 22 mm. Extensive spectral properties are listed on the Crystran website for each material, however for a brief overview summary please see below:

Window materialTransmission RangeRecommendations
Calcium fluoride

0.13 to 10 microns

Lower limit at beamline 1000 cm-1

Our most commonly used window material. Particularly good for cell culturing purposes or when working with wet samples, e.g. cyrosectioning, etc. Windows can be carefully washed and reused.
Barium fluoride

0.15 to 12 microns

Lower limit at beamline 900 cm-1

Lower wavenumber limit than CaF2 windows but the windows craze/go cloudy quickly when in the presence of water.
Zinc selenide

0.6 to 21 microns

Lower limit at beamline 750 cm-1 using the narrow-band MCT detector

Particularly good when working with wet samples or when analysis requires a lower wavenumber limit. Can be used in cell culturing, however this appears dependent on the cell line. Some lines easily adhere to the surface while others appear to show toxicity from the window material. It can help to first coat the windows in protein (soak in culture media) before cell culturing. Windows can be carefully washed and reused.


Where do you buy your IR transmissive windows

The company we typically purchase our IR transmissive windows from is Crystran, a company based in the UK. We normally recommend using their calcium fluoride windows, 0.5 mm thickness by 13 mm or 22 mm diameter. These are stock items so are typically delivered very quickly (we've had them within a week if the order is urgent). Zinc selenide and barium fluoride are also popular materials (0.5 mm thickness again), depending on the wavelength region of analysis. Some windows can be washed and reused (see Table above), however they do have a finite lifetime.

Can I wash and reuse my IR transmissive windows?

Yes, if the windows are not made of barium fluoride. Windows have a finite lifetime but can be washed and reused a limited number of times before becoming excessively scratched. Use water and/or a mild detergent to wash them, and lens tissue to gently polish them.

Can the beamline be used to examine soil samples?

Yes, the beamline has been used by several groups to examine soil particulates. If you require the spectra to be collected from individual microscopic sized grains or small particles of soil, this would be your primary argument in any proposal for your need to use synchrotron radiation. Remember however, that as the wavelength of mid-IR radiation is in the range 3-10 µm that this will be the approximate smallest sized objects that can be individually resolved.

Typical methods of sample preparation include using transmission mode by sandwiching the soil samples between diamond windows using our diamond window micro-compression cells. Many organic molecules are relatively strong absorbers of IR radiation so by flattening them between the windows, successful spectra of soil particulates can be collected. Water is however also a strong absorber and so you may need to consider whether you can dehydrate the soils without significantly disrupting the native chemistry. Some absorbed water can be worked with but it will mask spectral signatures coming from other O-H groups, C=O carboxylates etc., and to a lesser extent the C-O from carbohydrates etc.

Soil samples can also be embedded in an IR transmissive material like KBr. Previous groups have first gently pressed a thin KBr pellet (50 mg KBr, pressed at 7 tonnes for 2.5 minutes), then have sprinkled some particles selected with a fine needle on the top of the pellet before pressing again (7 tonnes for 2.5 minutes), to create thin pellets for transmission. A pellet press is available onsite for this purpose if required. An advantage of this method over use of the diamond window micro-compression cell is that the refractive index of KBr is close to that of soil, limiting any scattering effects around the edges of the particles. Disadvantages include a higher wavenumber cut-off closer to 900 cm-1, when compared to that achieved when using the compression cell with a cut-off closer to 750 cm-1.

How do I download my original data when offsite from the synchrotron?

At the completion of your beamtime, you will be sent an email with a link that allows you to access and download your data from the Australian Synchrotron SFTP service.  Follow the link and log in with you portal email address and password.  Once logged in, you can access a list of folders relating to each experiment you have attended.  You can download your data from here to your own local folder.  You will also receive a link with instructions on the secure file transfer protocol.

At the end of this email, you will also find a link to ownCloud to download a time limited version of OPUS 8.0, for analysing your spectral data.